The overall objective of this project is to study dynamic properties of neurite extension and changes in intracellular Ca2+ of adult rat dorsal root ganglion (DRG) neurons in culture. A combination of patch clamp and imaging techniques including confocal scanning laser microscopy and time- lapse video microscopy will be used for the studies. Neurite extension will be compared for neurons grown on polyornithine/laminin or feeder layers of astrocytes, Schwann cells, or oligodendrocytes to determine if neurite extension of adult DRG neurons is enhanced or hindered by presence of these glial cell types. Transmembrane fluxes and subcellular localization of Ca2+ in neurons loaded with the fluorescent dye fluo-3 will be studied in neurons during neurite extension to determine if the depolarization-dependent increases in intracellular Ca2+ we observe in the nucleus (and nucleolus) of DRG neurons is related to neurite extension. If these changes in the intranuclear Ca2+ precede neurite extension, this would suggest a possible role of Ca2+ in gene expression during neurite regeneration. Neurite extension and activity-dependent changes in intracellular Ca2+ will also be studied in DRG neurons that have been axotomize three weeks earlier. Our preliminary studies indicate more rapid and elaborate neurite extension in these pre-injured neurons: we will quantitate dynamic properties of neurite extension and changes in intracellular Ca2+ in these injured neurons. Ca2+ channel types, as defined by kinetics and pharmacology, will be studied with patch clamp and imaging techniques at various times during neurite extension to determine if Ca2+ channel types change during the course of neurite extension. Ca channels on neurites and growth cones will be studied with imaging techniques. The effects of impulse activity and Ca2+ channel blockade on neurite extension will be assessed. These studies will provide information on dynamic properties of neurite extension of adult mammalian neurons and how these responses are influenced by Ca2+, glial cells, and injury.